Transcript ppt presentation

Gene function analyses,
Reporter genes in direct and
reverse genetics
Reporter genes
• encode proteins that can be directly visualized or enzymes,
whose activity can be visualized
• quantitative or qualitative assessment
• promoter activity analyses, subcellular localization of
proteins, optimizing transformation procedure, ….
Constrains:
- background (autofluorescence, natural enzyme activity
within the tissue)
- protein stability (mask changes in promoter activity)
- degradation of fusion proteins
Reporter genes
gene
product
substrate
detection
gusA
b-glucuronidase (E. coli)
luc
gfp
luciferase (fire fly)
green fluorescent protein
(gelly fish)
MUG
X-gluc
luciferin
xxx
fluorescence
histochemical
luminiscence
fluorescence
Fluorescent proteins: GFP, DsRed, mCherry, EosFP
- unique tools for in vivo labelling
- encoded with small genes
Aequoria victoria
- origin: sea Coelenterata (corals, gelly fish)
- modified forms:
- fluorescent features,
- codon usage, splicing, stability, …
Barevné
varianty GFP
a DsRed
EosFP – photoactivatable fluorescent protein (green to red FP)
GUS
Qualitative detection (X-gluc)
• oxidized blue precipitate of reaction product
• low background
• slow diffusion
• mostly in fixed material
(X-gluc = 5-bromo-4-chloro-3-indolyl glucuronide)
Quantitative detection (MUG)
• GUS enzyme isolation, fluorimetric statement
• highly sensitive, low background
(MUG = 4-methylumbelliferyl-beta-D-glucuronide)
Gene function analyses
Modulation of expression:
- increased protein level (overexpression) –
introduction of a gene with a strong constitutive
promoter
- alt. gain-of-function mutations
- decreased protein level by RNAi
- alt. loss-off-function mutation
Tilling – point mutation in commercial collections
Reporter gene fusions
Decreasing protein level
Induction of RNA interference (dsRNA formation):
1) antisense RNA
2) hairpin RNA (e.g. sense-intron-antisense)
3) non-terminated RNA (dsRNA via RdRP)
Intermolecular pairing
+
intramolecular
pairing
RdRP
complementary strand
synthesis
- dsRNA cleavage by DCL, siRNA formation, sequence specific
mRNA degradation or block of transcription due to promoter
methylation
Promoter analysis
Fusion of analyzed promoter with reporter gene
(transcription fusion), with or without the original transcript:
reportérový gen
P
gen
T
- usually introduction of new copy into the genome
Indicates:
- tissue, organ, developmental specificity
- responses to external factors
Confirmation with other approaches advisible
(risk of artifacts)
Promoter fusion with GFP and GUS
Arabidopsis
thaliana
Fusion protein formation
- stop codon removal, fusion in reading frame
(= translational fusion)
- functional domains, natural interaction(!)
GFP fusion proteins
- Protein localization analyses, protein interactions
- in vivo labelling of cellular structures
Golgy complex
chromosomes
microtubules